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Isolation and Identification of EMT Subtypes.

Robert J Norgard1, Ben Z Stanger2,3,4

  • 1Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|September 17, 2020
PubMed
Summary

The epithelial-to-mesenchymal transition (EMT) drives cancer metastasis and chemoresistance. This study presents methods to isolate and identify epithelial, mesenchymal, and hybrid EMT cancer cell populations for further analysis.

Keywords:
E-cadherinEMTEpithelial-to-mesenchymal transitionFlow cytometry-activated cell sortingMagnetic-activated cell sortingPartial EMTPlasticitySubtypes

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Area of Science:

  • Oncology
  • Cell Biology
  • Cancer Research

Background:

  • Metastasis and chemoresistance are critical challenges in cancer, linked to the epithelial-to-mesenchymal transition (EMT).
  • EMT involves a spectrum of cellular phenotypes, including hybrid states, which impact tumor invasion and drug resistance.
  • Identifying EMT cancer cells in vivo is challenging due to their resemblance to stromal cells like cancer-associated fibroblasts.

Purpose of the Study:

  • To describe protocols for isolating epithelial and EMT cancer cell populations from murine tumors and cell cultures.
  • To enable the characterization of different EMT subtypes and their associated biological functions.
  • To present a flow cytometry method for identifying and studying tumor cells undergoing partial EMT.

Main Methods:

  • Two distinct methods for isolating epithelial and EMT cancer cell populations from primary murine tumors and cultured cancer cells.
  • Utilizing isolated populations for functional studies, gene expression analysis (RNA sequencing, RT-qPCR), DNA sequencing, epigenetic analysis, and protein analysis (western blotting, immunohistochemistry).
  • Employing a flow cytometry-based approach to identify and analyze tumor cells in partial epithelial-mesenchymal transition states.

Main Results:

  • The described protocols facilitate the isolation of distinct epithelial and EMT cancer cell populations.
  • These isolated cells serve as valuable tools for comprehensive downstream analyses, including molecular and functional characterization.
  • A flow cytometry method is established for identifying and studying tumor cells exhibiting partial EMT phenotypes.

Conclusions:

  • The presented protocols provide robust methods for isolating and characterizing diverse EMT cancer cell populations.
  • These methods are crucial for advancing our understanding of tumor cell plasticity, invasion, and drug resistance.
  • The identification and study of partial EMT states are essential for developing more effective cancer therapies.